End-stage organ failure or tissue loss is one of the most devastating and costly problems in medicine. The creation of engineered musculoskeletal tissue with functional myotendinous (MTJ) and neuromuscular (NMJ) junctions will not only restore the function of complex tissues such as muscle, tendon, and nerve following traumatic injury, but can also be used as a model for studying developmental muscle biology and muscle pharmacology. We have demonstrated that the co-culture of fetal nerve tissue with fetal or engineered tendon and engineered muscle tissue produces constructs with viable muscle-tendon interfaces that remain intact during force production, and viable neuromuscular junctions that advance the phenotype of the muscle tissue within the construct [1, 2]. The tissues formed express neonatal structures and do not substantially advance functionally or phenotypically in the absence of electrical or mechanical loading. The purpose of this study is to design, fabricate, and evaluate the structural and contractile characteristics of three-dimensional (3-D) engineered tissues containing myotendinous junctions (MTJ) and neuromuscular junctions (NMJ), two of the principal tissue interfaces required for a functional musculoskeletal construct. We propose to study the long-term viability of these constructs;to increase the expression of neurotrophic proteins like glial derived neurotrophic factor (GDNF) and introduce synthetic, engineered, and tissue based conduits to enhance innervation of the muscle constructs;to use bioreactors to place the nerve-tendon- muscle constructs into physical environments which simulate the stress, strain, and contractile activity resembling the mechanical milieu found in hind limb muscles in vivo;and to implant the constructs in vivo to surround the construct with the actual mechanical and biochemical environment of a hindlimb. Our working hypothesis is that an increase in the number of NMJs in conjunction with applied electrical activity in vitro will lead to a more advanced phenotype in the muscle and tendon, increased force production in the engineered muscles, and stronger and more developed tissue interfaces. In vivo implantation of in vitro engineered constructs will further advance the phenotype and functionality of all tissues and tissue interfaces. This project will address the following specific aims: Specific Aim 1. To design and fabricate nerve-tendon-muscle constructs with functional MTJs and NMJs and evaluate both the structural and functional integrity of the constructs at three time points of development. Specific Aim 2. To develop a conduit system to direct the growth of multiple neural extensions towards the muscle construct thus increasing the innervation ratio of the construct. Specific Aim 3. To evaluate the effect of muscle contraction in response to electrical stimulation on the structure and function of the nerve-tendon-muscle construct. Specific Aim 4. To evaluate the effect of an in vivo implantation into a host animal on the structure and function of the nerve-muscle tendon construct at four weeks.